The invention relates generally to the field of nucleic acid chemistry, and particularly to methods and compositions for labelling oligonucleotides on solid-supports. Label reagents include hybridization-stabilizing moieties, fluorescent dyes, fluorescence quenchers, energy-transfer dye sets, chemiluminescent dyes, metallo porphyrins, amino acids, proteins, peptides, enzymes, and affinity ligands.
Andrus, A. xe2x80x9cChemical methods for 5xe2x80x2 non-isotopic labelling of PCR probes and primersxe2x80x9d (1995) in PCR 2: A Practical Approach, Oxford University Press, Oxford, pp. 39-54.
Andrus, A., McCollum, C. and Zon, G. xe2x80x9cAutomated system for polynucleotide synthesis and purificationxe2x80x9d U.S. Pat. No. 5,047,524, issued Sep. 10, 1991.
Andrus, A., McCollum, C. and Zon, G. xe2x80x9cAutomated system for polynucleotide synthesis and purificationxe2x80x9d U.S. Pat. No. 5,262,530, issued Nov. 16, 1993.
Beaucage, S. and Iyer, R. xe2x80x9cAdvances in the synthesis of oligonucleotides by the phosphoramidite approachxe2x80x9d, Tetrahedron 48:2223-2311 (1992).
Bergot, B., Chakerian, V., Connell, C., Eadie, J., Fung, S., Hershey, N., Lee, L., Menchen, S. and Woo, S. xe2x80x9cSpectrally resolvable rhodamine dyes for nucleic acid sequence determinationxe2x80x9d, U.S. Pat. No. 5,366,860, issued Nov. 22, 1994.
Blackburn, G. and Gait, M. Eds. xe2x80x9cDNA and RNA structurexe2x80x9d in Nucleic Acids in Chemistry and Biology, 2nd Edition, (1996) Oxford University Press, pp. 15-81.
Bronstein, I. and Voyta, J., xe2x80x9cMethods of using chemiluminescent 1,2-dioxetanesxe2x80x9d U.S. Pat. No. 4,931,223, issued Jun. 5, 1990.
Bronstein, K., Fortin, J., Stanley, P., Stewart, G. and Kricka, L. xe2x80x9cChemiluminescent and bioluminescent reporter gene assaysxe2x80x9d, Anal. Biochemistry 219:169-81 (1994).
Cardullo, R., Agrawal, S., Flores, C., Zamecnik, P. and Wolf, D. xe2x80x9cDetection of nucleic acid hybridization by non-radiative fluorescence resonance energy transferxe2x80x9d, Proc. Natl. Acad. Sci. 85:8790-8794 (1988).
Caruthers, M. and Beaucage, S. xe2x80x9cPhosphoramidite compounds and processesxe2x80x9d, U.S. Pat. No. 4,415,732, issued Nov. 15, 1983.
Caruthers, M. and Matteucci, M. xe2x80x9cProcess for preparing polynucleotidesxe2x80x9d, U.S. Pat. No. 4,458,066, issued Jul. 3, 1984.
Clegg, R. xe2x80x9cFluorescence resonance energy transfer and nucleic acidsxe2x80x9d, Meth. Enzymol. 211:353-388 (1992).
Dib, C. Faure, S., Fizames, C., Samson, D., Drouot, N., Vignal, A., Millasseau, P., Marc, S., Hazan, J., Seboun, E., Lathrop, M., Gyapay, G., Morissette, J., Weissenbach J. xe2x80x9cA comprehensive genetic map of the human genome based on 5,264 microsatellitesxe2x80x9d, Nature 380:6570:152-4 (1996).
Dueholm, K., Egholm, M., Behrens, C., Christensen, L., Hansen, H., Vulpius, T., Petersen, K., Berg, R., Nielsen, P. and Buchardt, O. xe2x80x9cSynthesis of peptide nucleic acid monomers containing the four natural nucleobases: thymine, cytosine, adenine, and guanine and their oligomerizationxe2x80x9d, J. Org. Chem. 59:5767-73 (1994).
Egholm, M., Buchardt, O., Christensen, L., Behrens, C., Freier, S., Driver, D., Berg, R. and Kim, S. xe2x80x9cPNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen bonding rulesxe2x80x9d, Nature 365:566-68 (1993).
Englisch, U. and Gauss, D. xe2x80x9cChemically modified oligonucleotides as probes and inhibitorsxe2x80x9d, Angew. Chem. Int. Ed. Engl. 30:613-29 (1991).
Flanagan, W., Wagner, R., Grant, D., Lin, K. and Matteucci, M. xe2x80x9cCellular penetration and antisense activity by a phenoxazine-substituted heptanucleotidexe2x80x9d, Nature Biotech. 17:48-52 (1999).
Fodor, S., Pirrung, M., Read, J., and Stryer, L. xe2x80x9cArray of oligonucleotides on a solid substratexe2x80x9d, U.S. Pat. No. 5,445,934, issued Aug. 29, 1995.
Gong, B. and Yan, Y. xe2x80x9cNew DNA minor-groove binding molecules with high sequence-selectivities and binding affinitiesxe2x80x9d, Biochem. and Biophys. Res. Comm. 240:557-60 (1997).
Grossman, P., Bloch, W., Brinson, E., Chang, C., Eggerding, F., Fung, S., Iovannisci, D., Woo, S. and Winn-Dean, E. xe2x80x9cHigh-density multiplex detection of nucleic acid sequences: oligonucleotide ligation assay and sequence-coded separationxe2x80x9d, Nucl. Acids Res. 22:4527-4534 (1994).
Hermanson, G. in Bioconjugate Techniqiies (1996) Academic Press, San Diego, pp. 40-55, 643-671.
Ju, J., Kheterpal, I., Scherer, J., Ruan, C., Fuller, C., Glazer, A. and Mathies, R. xe2x80x9cDesign and Synthesis of fluorescence energy transfer dye-labeled primers and their application for DNA sequencing and analysisxe2x80x9d, Analytical Biochemistry 231:131-140 (1995).
Keller, G. and Manak, M. in DNA Probes Second Edition (1993), Stockton Press, New York, pp. 121-23.
Kricka, L. in Nonisotopic DNA Probe Techniquies (1992), Academic Press, San Diego, pp. 3-28.
Kubista, M. and Svanvik, N. xe2x80x9cProbe for analysis of nucleic acidsxe2x80x9d, WO 97/45539, Intl. Publ. Date Dec. 4, 1997.
Kutyavin, I., Lukhtanov, E., Gamper, H. and Meyer, R. xe2x80x9cCovalently linked oligonucleotide minor groove binder conjugatesxe2x80x9d, WO 96/32496, Intl. Publ. Date Oct. 17, 1996.
Lee, L., Spurgeon, S., Rosenblum, B. xe2x80x9cEnergy transfer dyes with enhanced fluorescencexe2x80x9d, U.S. Pat. No. 5,800,996, issued Sep. 1, 1998.
Lee, L., Spurgeon, S., Heiner, C., Benson, S., Rosenblum, B., Menchen, S., Graham, R., Constantinescu, A., Upadhya, K and Cassel, M. xe2x80x9cNew energy transfer dyes for DNA sequencingxe2x80x9d, Nucl. Acids Res. 25:2816-22 (1997).
Livak, K., Flood, S., Marmaro, J., Giusti, W. and Deetz, K. xe2x80x9cOligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridizationxe2x80x9d, PCR Methods and Applications 4:357-362 (1995).
Livak, K., Flood, S. and Marmaro, J. xe2x80x9cMethod for Detecting Nucleic Acid Amplification Using Self-Quenching Fluorescence Probexe2x80x9d, U.S. Pat. No. 5,538,848, issued Jul. 23, 1996.
Livak, K., Flood, S., Marmaro, J. and Mullah, K. xe2x80x9cSelf-quenching fluorescence probexe2x80x9d, U.S. Pat. No. 5,723,591, issued Mar. 3, 1998.
Lukhtanov, E., Kutyavin, I., Gamper, H. and Meyer, R. xe2x80x9cOligodeoxyribonucleotides with conjugated dihydropyrroloindole oligopeptides: Preparation and hybridization propertiesxe2x80x9d, Bioconjugate Chem. 6:418-26 (1995).
Menchen, S., Lee, L., Connell, C., Hershey, N., Chakerian, V., Woo, S. and Fung, S. xe2x80x9c4,7-Dichlorofluorescein dyes as molecular probesxe2x80x9d, U.S. Pat. No. 5,188,934, issued Feb. 23, 1993.
Meyer, R. xe2x80x9cIncorporation of modified bases in oligonucleotidesxe2x80x9d in Protocols for Oligonucleotide Conjugates, Ed. S. Agrawal (1994) Humana Press, Totowa, N.J., pp. 73-92.
Mullah, B. and Andrus, A. xe2x80x9cAutomated synthesis of double dye-labeled oligonucleotides using tetramethylrhodamine (TAMRA) solid supportsxe2x80x9d, Tetrahedron Letters 38: 5751-5754 (1997).
Mullah, B. and Andrus, A. xe2x80x9cSolid support reagents for the direct synthesis of 3xe2x80x2-labeled polynucleotidesxe2x80x9d, U.S. Pat. No. 5,736,626, issued Apr. 7, 1998.
Mullah, B., Livak, K., Andrus, A. and Kenney, P. xe2x80x9cEfficient synthesis of double dye-labeled oligodeoxyribonucleotide probes and their application in a real time PCR assayxe2x80x9d, Nucl. Acids Res. 26:1026-1031 (1998).
Nelson, P., Kent, M. and Muthini, S. xe2x80x9cOligonucleotide labeling methods 3. Direct labeling of oligonucleotides employing a novel, non-nucleosidic, 2-aminobutyl-1,3-propanediol backbonexe2x80x9d, Nucl. Acids Res. 20:6253-59 (1992).
Nelson, P. xe2x80x9cMultifunctional controlled pore glass reagent for solid phase oligonucleotide synthesisxe2x80x9d, U.S. Pat. No. 5,141,813, issued Aug. 25, 1992.
Nielsen, P., Egholm, M., Berg, R. and Buchardt, O. xe2x80x9cSequence-selective recognition of DNA by strand displacement with a thymidine-substituted polyamidexe2x80x9d, Science 254:1497-1500 (1991).
Stanton, T., Schindele, D., Renzoni, G., Pepich, B., Anderson, N., Clagett, J. and Opheim, K. xe2x80x9cPreparation and use of monomeric phthalocyanine reagentsxe2x80x9d WO 8804777, Intl. Publ. Date: Jun. 30, 1988.
Theisen, P., McCollum, C. and Andrus, A. xe2x80x9cFluorescent dye phosphoramidite labelling of oligonucleotidesxe2x80x9d, in Nucleic Acid Symposium Series No. 27, Oxford University Press, Oxford, pp. 99-100 (1992).
Tyagi, S. and Kramer, F. xe2x80x9cMolecular Beacons: Probes that fluoresce upon hybridizationxe2x80x9d, Nature Biotechnology, 14:303-08 (1996).
Van der Laan, A., Brill, R., Kuimelis, R., Kuyl-Yeheskiely, E., van Boom, J., Andrus, A. and Vinayak, R. xe2x80x9cA convenient automated solid-phase synthesis of PNA-(5xe2x80x2)-DNA-(3xe2x80x2)-PNA chimeraxe2x80x9d, Tetrahedron Lett. 38:2249-52 (1997).
Vinayak, R., van der Laan, A., Brill, R., Otteson, K., Andrus, A., Kuyl-Yeheskiely, E. and van Boom, J. xe2x80x9cAutomated chemical synthesis of PNA-DNA chimera on a nucleic synthesizerxe2x80x9d, Nucleosides and Nucleotides 16:1653-56 (1997).
Wagner, T. and Pfleiderer, W. xe2x80x9cAn inverse approach in oligodeoxyribonucleotide synthesisxe2x80x9d, Nucleosides and Nucleotides 16:1657-60 (1997).
Woo, S., Menchen, S. and Fung, S. xe2x80x9cRhodamine phosphoramidite compoundsxe2x80x9d, U.S. Pat. No. 5,231,191, issued Jul. 27, 1993.
Woo, S. and Fung, S. xe2x80x9cSolid support reagents for the synthesis of 3xe2x80x2-nitrogen containing polynucleotidesxe2x80x9d, U.S. Pat. No. 5,552,471, issued Sep. 3, 1996.
Non-isotopically labelled oligonucleotides are essential components in many important molecular biology applications, such as PCR amplification, DNA sequencing, antisense transcriptional and translational control of gene expression, genetic analysis, and DNA probe-based diagnostic testing (Keller, 1993; Kricka, 1992). Fluorescence detection of fluorescent dye-labelled oligonucleotides is the basis for nucleic acid sequence detection assays such as Taqman(trademark) (Livak, 1996), Molecular Beacons (Tyagi, 1996), genetic linkage mapping (Dib, 1996), and oligonucleotide-ligation assay (Grossman, 1994).
Two general methods for labeling synthetic oligonucleotides have been established. In a first method, referred to herein as the xe2x80x9ctwo-step solution labelling methodxe2x80x9d, a nucleophilic functionality, e.g. a primary aliphatic amine, is introduced at a labelling attachment site on an oligonucleotide, e.g. a 5xe2x80x2 terminus. After automated, solid-support synthesis is complete, the oligonucleotide is cleaved from the support and all protecting groups are removed. The nucleophile-oligonucleotide is reacted with an excess of a label reagent containing an electrophilic moiety, e.g. isothiocyanate or activated ester, e.g. N-hydroxysuccinimide (NHS), under homogeneous solution conditions (Hermanson, 1996; Andrus, 1995).
In a second alternative method, referred to herein as the xe2x80x9cdirect labeling methodxe2x80x9d, a label is directly incorporated into the oligonucleotide during or prior to synthesis (Mullah, 1998; Nelson, 1992). The direct labelling method is preferred because it (i) does not require a post-synthesis reaction step, thereby simplifying the synthesis of labelled polynucleotides; and (ii) avoids the problems associated with the low reaction yield ( less than 60%) typically encountered with the two-step solution labelling method, namely: (a) purification of the labeled oligonucleotide from excess label; (b) purification of the labeled oligonucleotide from unlabeled oligonucleotide; (c) high costs due to the low product yield and laborious analytical and purification procedures, and; (d) irreversible capping of the nucleophilic functionality during synthesis.
Certain fluorescent dyes and other labels have been functionalized as phosphoramidite reagents for 5xe2x80x2 labelling (Theisen, 1992). However, some labels, e.g., digoxigenin, rhodamine dyes, and cyanine dyes, are too unstable to survive the harsh conditions and reagents used in reagent preparation and oligonucleotide synthesis, cleavage and deprotection. Thus, whenever such labels are used in current solid phase synthesis protocols, they must be attached using the less preferred two-step solution labelling method.
Therefore it is desirable to provide methods and reagents to label oligonucleotides and analogs directly on a solid-support upon which they are synthesized, under conditions which are rapid, economical, and compatible with chemically-labile functionality.
The present invention is directed toward novel methods and compositions for synthesis of labelled oligonucleotides on solid-supports.
In a first aspect, the invention comprises a method for synthesis of labelled oligonucleotides on a labelled solid-support having the structure 
where S is a solid-support, A is a cleavable linker, X is a moiety with three or more attachment sites, L is a label, Y is a nucleophile, i.e. O, NH, NR or S, and P1 is an acid cleavable protecting group. The labelled solid-support is reacted in a cyclical fashion with reagents to: (1) remove P1 from Y, (2) couple Y with the 5xe2x80x2 position of a 5xe2x80x2-phosphoramidite, 3xe2x80x2 protected nucleoside, (3) cap any unreacted sites on the support, e.g. Y, if necessary, and (4) oxidize any phosphite linkages. The four steps are repeated until the entire labelled oligonucleotide is synthesized.
After synthesis is complete, protecting groups on the internucleotide phosphates and nucleobases of the labelled oligonucleotide may be removed by deprotection while the oligonucleotide remains on the solid-support. Alternatively, after synthesis is complete, the labelled oligonucleotide may be cleaved from the solid-support and then deprotected.
In a second aspect, the invention comprises a nucleoside bound to a solid-support having the structure 
where S, A, X, L, and Y are defined as before, R is a phosphate protecting group or phosphotriester substituent; B is a nucleobase; P2 is an exocyclic nitrogen protecting group; and P3 is an acid-labile protecting group.
In a third aspect, the invention comprises an oligonucleotide bound to a solid-support having the structure 
where the variable substituents are defined as before, and n is an integer preferably from about 0 to 100.
In a fourth aspect, the invention comprises a method for synthesizing a labelled oligonucleotide by reacting: (i) a label reagent bearing functionality that can be converted into an electrophile, e.g. carboxylic acid, sulfonic acid, phosphonic acid, or phosphoric acid, (ii) an oligonucleotide on a solid support with a nucleophilic functionality, e.g. alcohol, amine, or thiol, and (iii) a coupling reagent, whereby an ester, amide, thioester, sulfonamide, sulfonate, phosphonate, phosphoramidate, phosphorothioate, or phosphate bond is formed. The labelling method may be conducted on an oligonucleotide at label sites including the 5xe2x80x2 terminus, the 3xe2x80x2 terminus, a nucleobase, an internucleotide linkage, a sugar, amino, sulfide, hydroxyl, and carboxyl. The labelling reaction may be conducted on an oligonucleotide comprising one or more DNA, RNA, PNA and nucleic acid analog monomer units. The nucleic acid analogs may be nucleobase, sugar, and/or internucleotide analogs.
The labelled oligonucleotide may be synthesized either in the 5xe2x80x2 to 3xe2x80x2 direction, or in the 3xe2x80x2 to 5xe2x80x2 direction.